strain: B6C3F1 developmental stage: adult gender: male tissue: lung treatment group: Control
Treatment protocol
Age matched adult male B6C3F1 mice (27-30 d, Charles Rivers Laboratories, St Constant, Quebec, Canada) were housed individually under a 12:12 h light: dark cycle with food and water available ad libitum. Mice were randomly assigned (6 per group) to a control or treatment group. Mice were treated with a daily dose of BaP in corn oil with 5, 50, 150 or 300 mg/kg (oral gavage, 10 ml/kg) for three consecutive days. Control mice received corn oil only. Mice were anaesthetized under isofluorane and sacrificed by exsanguination 4 or 24 hours after the last treatment. Right and left lung lobes were removed and immediately snap frozen and stored at -80ºC until use. Blood serum was collected as described below. All animal procedures were in accordance with the guidelines of the Canadian Council for Animal Care and approved by the Health Canada Animal Care Committee.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from random sections of the left lung using TRIzol reagent (Invitrogen) and purified using RNeasy Mini Kit (Qiagen). All RNA samples showing A260/280 ratios between 2.0 and 2.1 were further analysed for RNA integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). Only high quality RNA (28S/18S > 1.8) was used for analysis. Was quantified using UV spectrophotometer and quality was confirmed using the Agilent 2100 Bioanalyzer and RNA 6000 NanoLab Chip Kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada).
Label
Cy5
Label protocol
Global mRNA profiling was conducted on 5 control samples alongside 5 mice exposed to 150 mg/kg and 5 mice exposed to 300 mg/kg BaP from the 4 hour time point for both liver and lung. Individual total RNA (250 ng) samples and universal reference total RNA (Stratagene) were used to synthesize double-stranded cDNA and cyanine labelled cRNA according to the manufacturer’s instructions (Agilent Linear Amplification Kits, Agilent Technologies). Experimental samples were labelled with Cyanine 5-CTP, and reference RNA with Cyanine 3-CTP (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada). Cyanine-labelled cRNA targets were in vitro transcribed using T7 RNA polymerase and purified by RNeasy Mini Kit (Qiagen). Labelled cRNA was hybridized to Agilent mouse 4 x 44 oligonucleotide microarrays (Agilent Technologies) at 60ºC overnight. Arrays were washed and scanned on an Agilent G2505B scanner at 5 m resolution. Data were acquired using Agilent Feature Extraction software version 9.5.3.1.
sample: universal mouse reference total RNA (Stratagene)
Treatment protocol
Age matched adult male B6C3F1 mice (27-30 d, Charles Rivers Laboratories, St Constant, Quebec, Canada) were housed individually under a 12:12 h light: dark cycle with food and water available ad libitum. Mice were randomly assigned (6 per group) to a control or treatment group. Mice were treated with a daily dose of BaP in corn oil with 5, 50, 150 or 300 mg/kg (oral gavage, 10 ml/kg) for three consecutive days. Control mice received corn oil only. Mice were anaesthetized under isofluorane and sacrificed by exsanguination 4 or 24 hours after the last treatment. Right and left lung lobes were removed and immediately snap frozen and stored at -80ºC until use. Blood serum was collected as described below. All animal procedures were in accordance with the guidelines of the Canadian Council for Animal Care and approved by the Health Canada Animal Care Committee.
Extracted molecule
total RNA
Extraction protocol
Unknown: Material was purchased as total RNA (universal mouse reference total RNA (Stratagene, CA, USA))
Label
Cy3
Label protocol
Global mRNA profiling was conducted on 5 control samples alongside 5 mice exposed to 150 mg/kg and 5 mice exposed to 300 mg/kg BaP from the 4 hour time point for both liver and lung. Individual total RNA (250 ng) samples and universal reference total RNA (Stratagene) were used to synthesize double-stranded cDNA and cyanine labelled cRNA according to the manufacturer’s instructions (Agilent Linear Amplification Kits, Agilent Technologies). Experimental samples were labelled with Cyanine 5-CTP, and reference RNA with Cyanine 3-CTP (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada). Cyanine-labelled cRNA targets were in vitro transcribed using T7 RNA polymerase and purified by RNeasy Mini Kit (Qiagen). Labelled cRNA was hybridized to Agilent mouse 4 x 44 oligonucleotide microarrays (Agilent Technologies) at 60ºC overnight. Arrays were washed and scanned on an Agilent G2505B scanner at 5 m resolution. Data were acquired using Agilent Feature Extraction software version 9.5.3.1.
Hybridization protocol
Global mRNA profiling was conducted on 5 control samples alongside 5 mice exposed to 150 mg/kg and 5 mice exposed to 300 mg/kg BaP from the 4 hour time point for both liver and lung. Individual total RNA (250 ng) samples and universal reference total RNA (Stratagene) were used to synthesize double-stranded cDNA and cyanine labelled cRNA according to the manufacturer’s instructions (Agilent Linear Amplification Kits, Agilent Technologies). Experimental samples were labelled with Cyanine 5-CTP, and reference RNA with Cyanine 3-CTP (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada). Cyanine-labelled cRNA targets were in vitro transcribed using T7 RNA polymerase and purified by RNeasy Mini Kit (Qiagen). Labelled cRNA was hybridized to Agilent mouse 4 x 44 oligonucleotide microarrays (Agilent Technologies) at 60ºC overnight. Arrays were washed and scanned on an Agilent G2505B scanner at 5 m resolution. Data were acquired using Agilent Feature Extraction software version 9.5.3.1.
Scan protocol
Global mRNA profiling was conducted on 5 control samples alongside 5 mice exposed to 150 mg/kg and 5 mice exposed to 300 mg/kg BaP from the 4 hour time point for both liver and lung. Individual total RNA (250 ng) samples and universal reference total RNA (Stratagene) were used to synthesize double-stranded cDNA and cyanine labelled cRNA according to the manufacturer’s instructions (Agilent Linear Amplification Kits, Agilent Technologies). Experimental samples were labelled with Cyanine 5-CTP, and reference RNA with Cyanine 3-CTP (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada). Cyanine-labelled cRNA targets were in vitro transcribed using T7 RNA polymerase and purified by RNeasy Mini Kit (Qiagen). Labelled cRNA was hybridized to Agilent mouse 4 x 44 oligonucleotide microarrays (Agilent Technologies) at 60ºC overnight. Arrays were washed and scanned on an Agilent G2505B scanner at 5 m resolution. Data were acquired using Agilent Feature Extraction software version 9.5.3.1.
Data processing
A reference design [50, 51] was used to analyse gene expression microarray data. The design was blocked on the slide, since the Agilent arrays contain 4 arrays per slide. The background fluorescence was measured using the (-)3xSLv1 probes; probes with median signal intensities less than the trimmed mean (trim = 5%) plus three trimmed standard deviations of the (-)3xSLv1 probe were flagged as absent (within the background signal). Data were normalized using LOWESS in R [52]. Ratio intensity plots and heat maps for the raw and normalized data were constructed to identify outliers. One sample was removed from the analysis based on clustering. Genes that were differentially expressed as a result of treatment were determined using the MAANOVA library in R [53]. The main effect in the model was treatment. This model was applied to the log2 of the absolute intensities. The Fs statistic [54] was used to test for treatment effects. The p-values for all statistical tests were estimated by the permutation method using residual shuffling, followed by adjustment for multiple comparisons using the false discovery rate (FDR) approach [55]. The fold change calculations were based on the least-square means. Significant genes identified as having an adjusted p-value < 0.05 for any individual contrast. RawCy5 and RawCy3 are the median signal intensities for the Cy5 and Cy3 channels on each microarray. NormCy5 and NormCy3 are the loess normalized signal intensities for the Cy5 and Cy3 channels within each array; this is a within-array normalization performed separately for each array. Flag indicates whether the median signal intensity falls in the background, i.e below threshold calculated from the negative controls (mean plus three standard deviations of the negative controls.) Ratio is calculated as log2(NormCy5/NormCy3)
